Migraine is the second leading cause of disability worldwide; however, it remains unclear how the headache phase is initiated during a migraine attack. In migraine with aura, a condition that affects about 30% of people with migraine, the leading theory proposes that (a) cortical spreading depression (CSD) is the pathophysiological event that underlies the aura phase, and b) CSD somehow leads to the activation of the meningeal sensory system, resulting in the onset of the headache. Our recent studies, including during the prior grant period, provided a long-missing critical piece of support for the theory by finally showing that CSD does in fact activate meningeal sensory afferents, and can produce a prolonged period of both activation and mechanical sensitization. However, a long-standing problem with the CSD theory has been the paucity of evidence that CSD leads to pain behaviors, as would be expected for an event that supposedly is a potent trigger for headache. Furthermore, efforts to establish a link between CSD, the ensuing meningeal afferent responses, and headache pain have been limited by the challenges of studying the activity of meningeal sensory neurons in awake behaving animals. We have overcome this major obstacle by developing a two- photon calcium imaging approach to monitor the activity of meningeal afferent nerve endings in the awake behaving mouse, during voluntary locomotion via a chronic cranial window. In Aim 1, we propose to pursue this novel imaging approach to expand upon preliminary data suggesting that as a result of CSD-induced mechanical sensitization, meningeal stretching during voluntary locomotion results in enhanced activation of meningeal afferents, and consequent reduction of locomotion. Aims 2 and 3 are to explore cellular and molecular mechanisms that contribute to meningeal nociception following CSD. We will build upon preliminary results and use electrophysiology in anesthetized animals, calcium imaging in awake mice and an optogenetic approach to test the hypothesis that cortical astrocytes play a causal role in mediating the enhanced meningeal afferent responses and related decrease in voluntary locomotion following CSD. We will then employ biosensors, electrophysiology, afferent calcium imaging, pharmacological inhibitors and transgenic mice to test the hypothesis that astrocyte-dependent cortical ATP efflux contributes to CSD-evoked meningeal nociception via purinergic P2X7 signaling in meningeal macrophages. These innovative experiments, in addition of establishing a novel powerful platform for studying the responses of meningeal afferents and associated behavioral consequences related to migraine headache, could also shed light on the roles of cortical astrocytes and meningeal purinergic signaling, as well as sex differences in the mechanisms responsible for migraine pain - a key step towards mitigating the painful effects of CSD in migraine with aura.